The Effects of Phosphorylation of Cysteine String Protein on Retinal Waves

視覺系統在發育的關鍵時期會產生一種自發性的放電現象，稱為「視網膜波」，對於神經迴路的正確連結極為重要。視網膜波的形成是透過突觸前神經元—星狀無軸突細胞—釋放乙醯膽鹼，經由突觸後神經元—視網膜節細胞—接收後而產生。先前的研究顯示，發育中的星狀無軸突細胞能週期性地產生神經衝動並進行鈣離子調控的胞吐作用，釋放興奮性神經傳導物質例如乙醯膽鹼和γ-丁氨基酪酸，進而引發視網膜波。然而，其中仍不甚了解的是，星狀無軸突細胞中鈣離子調控之胞吐作用的改變如何能影響視網膜波的時空特性。 半胱胺酸串鍊蛋白被發現能幫助SNARE蛋白進行正確摺疊，SNARE蛋白已被證實為胞吐作用中膜融合的參與分子，因此半胱胺酸串鍊蛋白可藉由影響SNARE蛋白來調控神經傳導物質的釋放。此外，半胱胺酸串鍊蛋白能被蛋白質激酶A所磷酸化，而這種蛋白質激酶在視網膜波發生的時期會被高度活化。這些結果暗示，星狀無軸突細胞或許能經由細胞內訊息傳遞路徑以調控半胱胺酸串鍊蛋白的磷酸化及其功能，進而影響視網膜波的時空特性。 在此論文中，我們研究半胱胺酸串鍊蛋白的磷酸化是否能影響視網膜波的時空特性。首先，我們使用免疫螢光染色證明半胱胺酸串鍊蛋白能在新生老鼠的內網狀層中表現，在分離的視網膜細胞中，更發現半胱胺酸串鍊蛋白能表現在星狀無軸突細胞內，說明了半胱胺酸串鍊蛋白存在於星狀無軸突細胞中，可能可以調控其神經傳導物質釋放的機制。此外，定量聚合酶鏈鎖反應的實驗結果顯示，發育中的視網膜內最主要的半胱胺酸串鍊蛋白為α型。為了更進一步研究突觸前星狀無軸突細胞中半胱胺酸串鍊蛋白的磷酸化是否會影響視網膜波，我們將特定基因（半胱胺酸串鍊蛋白或其磷酸化突變株）利用專一性啟動子大量地表現於星狀無軸突細胞之中，再利用鈣離子顯像技術記錄視網膜節細胞層的鈣離子變化以偵測視網膜波的時空特性。我們發現當星狀無軸突細胞表現半胱胺酸串鍊蛋白磷酸化缺陷型S10A時，會顯著降低視網膜波的發生頻率；其餘控制組、半胱胺酸串鍊蛋白野生型及磷酸化模擬型S10D & S10E則否。相較之下，半胱胺酸串鍊蛋白對於視網膜的空間性質只有較小的影響。全細胞膜電壓固定實驗顯示，半胱胺酸串鍊蛋白磷酸化缺陷型S10A降低視網膜節細胞內突觸後電流頻率和其斜率，但不影響視網膜節細胞的電生理特質，顯示大量表現磷酸化缺陷型S10A所造成的結果是來自突觸前星狀無軸突細胞中神經傳導物質釋放的改變所致。有鑑於半胱胺酸串鍊蛋白磷酸化缺陷型S10A會降低視網膜波的頻率而不影響其空間性質，我們的研究顯示半胱胺酸串鍊蛋白的磷酸化，在調控視網膜波的時間性質上，扮演著重要角色。During a developmental critical period, the visual system displays a robust spontaneous activity termed “retinal waves”, essential for neural circuit refinement. These waves are initiated by releasing neurotransmitters from presynaptic starburst amacrine cells (SACs) onto postsynaptic retinal ganglion cells (RGCs). Previous studies showed that the developing SACs can periodically fire action potentials and undergo Ca2+-regulated exocytosis, thus releasing excitatory neurotransmitters such as acetylcholine (ACh) and γ-amino butyric acid (GABA) and inducing periodic retinal waves. However, little is known regarding how altering the Ca2+-regulated exocytosis in SACs affects the spatial or temporal properties of retinal waves. Cysteine string protein (CSP) was found to ensure the correct folding of fusion machinery [i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins], thus playing an important role in regulating neurotransmitter release. In addition, CSP can be phosphorylated by protein kinase A (PKA) that is highly activated during retinal waves. These results suggest that the intracellular signaling in SACs may modulate the function of CSP and thus regulate neurotransmitter release during retinal waves. In this study, we investigated how phosphorylation of CSP affects the spatial or temporal properties of retinal waves. First, we used immunofluorescence staining to show that CSP was expressed in the inner plexiform layer (IPL) of the neonatal rat retina. Further immunostaining of dissociated retinal cells confirmed that the expression of CSP was localized to presynaptic SACs, implying that CSP may involve in regulating neurotransmitter release from SACs. The quantitative polymerase chain reaction (qPCR) experiment showed that CSPα was the dominant isoform in the developing rat retina. To investigate whether phosphorylation of CSPα in presynaptic SACs affects retinal waves, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter (pmGluR2). After overexpression of CSP or its phosphomutants in SACs, subsequent Ca2+ imaging was performed in the RGC layer to detect the spatial and temporal properties of wave-associated spontaneous Ca2+ transients. We found that the frequency of Ca2+ transients was significantly decreased by the phosphodeficient mutant (CSPα-S10A), but not by the wild-type CSPα (CSPα-WT) or the phosphomimetic mutants (CSPα-S10D and CSPα-S10E) compared to the control. In contrast, the CSPα phosphodeficient mutant had a relatively minor effect on the spatial correlation of spontaneous Ca2+ transients over distance. Whole-cell voltage-clamp recordings demonstrated that the CSPα phosphodeficient mutant reduced the frequency and the slope of wave-associated postsynaptic currents (PSCs), but not altered the electrical properties of postsynaptic RGCs, suggesting that the effects by overexpressing the CSPα phosphodeficient mutant were due to the change in the release from presynaptic SACs. Given that the CSPα phosphodeficient mutant down-regulates the frequency but does not significantly alter the spatial correlation of retinal waves, our results suggest that phosphorylation of CSPα may play an important role in regulating the temporal properties of retinal waves.國立臺灣大學碩士學位論文口試委員會審定書......................................................... i Acknowledgements ........................................................................................................ ii 中文摘要....................................................................................................................... iv Abstract ......................................................................................................................... vi Abbreviations ................................................................................................................ ix Contents ...................................................................................................................... xiii Chapter I Introduction .................................................................................................................... 1 1.1 Development of the nervous system ................................................................. 1 1.2 Organization of mammalian visual system ...................................................... 1 1.3 The structure of retina ...................................................................................... 2 1.4 Visual phototransduction pathway ................................................................... 2 1.5 Retinal development ......................................................................................... 4 1.6 Retinal waves.................................................................................................... 4 1.7 Cysteine string protein (CSP) ........................................................................... 7 1.8 Specific aims .................................................................................................. 10 Chapter II Materials and Methods ............................................................................................. 12 2.1 Animals........................................................................................................... 12 2.2 Plasmid construction and subcloning ............................................................. 12 2.3 Dissection of retinas and optic nerves ............................................................ 15 2.4 Retinal explants culture .................................................................................. 16 2.5 Transient transfection ..................................................................................... 17 2.6 Immunofluorochemistry ................................................................................. 18 2.7 Immunofluorescence staining for dissociated retinal cells ............................. 20 2.8 Antibodies....................................................................................................... 21 2.9 Calcium imaging ............................................................................................ 21 2.10 Data analysis of spontaneous Ca2+ transients ............................................... 23 2.11 Retinal RNA extraction ................................................................................ 24 2.12 Primary retinal cell culture ........................................................................... 26 2.13 Whole-cell patch-clamp recordings .............................................................. 27 Chapter III Results ...................................................................................................................... 29 3.1 CSP was expressed in the developing rat retina ............................................. 29 3.2 CSP was expressed in the optic nerves (the axons of retinal ganglion cells) . 30 3.3 CSPα1 was the major CSP isoform in the developing rat retina .................... 30 3.4 The frequency of Ca2+ transients in postsynaptic RGCs were significantly decreased by overexpressing the phosphodeficient mutant CSP-S10A in presynaptic SACs ................................................................................................. 31 3.5 Spatial properties of retinal waves were not changed by overexpressing CSP-S10A in presynaptic SACs ........................................................................... 32 3.6 The frequency of wave-associated postsynaptic currents (PSCs) in RGCs was significantly decreased by overexpressing CSP-S10A in presynaptic SACs ....... 33 3.7 The slope of wave-associated PSCs was decreased by overexpressing CSP-S10A in presynaptic SACs ........................................................................... 35 3.8 The intrinsic membrane properties of RGCs were not changed by overexpressing CSP-S10A in presynaptic SACs ................................................. 36 Chapter IV Discussion ................................................................................................................ 37 4.1 Endogenous CSP in the developing rat retina ................................................ 37 4.2 Presynaptic CSP in regulating retinal waves .................................................. 38 4.3 CSP regulates the releasing properties in presynaptic SACs ......................... 40 4.4 Pros and cons of the study .............................................................................. 41 4.5 Future directions ............................................................................................. 44 Chapter V Conclusion ................................................................................................................ 46 References .................................................................................................................... 47 List of Figures Figure 1. The structure of vertebrate eyeball and retina ........................................... 52 Figure 2. The central projection targets of retinal ganglion cells (RGCs) ............... 53 Figure 3. The retinal waves and the underlying mechanisms .................................. 54 Figure 4. Retinal waves drive the activation of protein kinase A (PKA), which also plays a role in regulating retinal waves .................................................................... 55 Figure 5. The structure and proposed function of cysteine string protein (CSP) ..... 56 Figure 6. Ca2+-regulated exocytosis modulated by CSP phosphorylation ............... 57 Figure 7. CSP was expressed around the rat IPL during development and partially colocalized to SACs ................................................................................................. 58 Figure 8. CSP was expressed in the developing rat retina ....................................... 59 Figure 9. CSP was expressed in the developing rat SACs ....................................... 60 Figure 10. Colocalization ratios between CSP and ChAT immunoreactivity signals ................................................................................................................................. 61 Figure 11. CSP was expressed in the developing rat optic nerves ........................... 62 Figure 12. The mRNA levels of CSP isoforms in the developing rat retina ............ 63 Figure 13. Transfected SACs, Fura-2-loaded RGCs and the recordings of spontaneous Ca2+ transients after ex vivo retinal transfection .................................. 64 Figure 14. The frequency and interval of spontaneous Ca2+ transients were changed by overexpression of CSP-S10A in SACs ............................................................... 66 Figure 15. The amplitude and duration of spontaneous Ca2+ transients were not changed by overexpression of wild-type or mutant CSP in SACs ........................... 68 Figure 16. The spatial correlation of spontaneous Ca2+ transients ........................... 70 Figure 17. The frequency of postsynaptic currents (PSCs) in RGCs were changed by overexpression of CSP-S10A in SACs ............................................................... 71 Figure 18. The slope to the peak PSC was decreased by overexpression of CSP-S10A in SACs .................................................................................................. 73 Figure 19. The membrane properties of RGCs were not changed by overexpression of CSP-S10A in SACs .............................................................................................. 75 List of Tables Table 1. The values of correlation indexes from each construct (Ctrl, CSP-WT, CSP-S10A, CSP-S10D, and CSP-S10E) .................................................................. 76 Table 2. Antibodies used for immunofluorescence staining .................................... 78 Appendix The 9th International Symposium of the Kanagawa University - National Taiwan University Exchange Program: Abstract and Poster ................................................ 80 2014 Institute of Molecular and Cellular Biology Poster Contest: Poster ............... 82 Figure for PLoS One Paper “Adenosine A2A Receptor Up-Regulates Retinal Wave Frequency via Starburst Amacrine Cells in the Developing Rat Retina” (Huang et al., 2014): The immunofluorescence staining of A2AR in the developing rat SACs ................................................................................................................................. 8